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Preparation of extracts: Coral samples of orange and red colonies of Montipora capitata were collected in Kaneohe Bay, Oahu. Frozen samples were extracted in 2:1 dichloromethane:methanol, which was replaced daily for 3 d. Extracts were filtered, dried, and weighed. We tested extracts at concentrations approximating those naturally found in the coral tissues (Gochfeld & Aeby 2008). To determine the volumetric concentrations of extracts, the surface area of each piece of coral was calculated using the wax technique (Gochfeld 1991). Tissue volume was determined by multiplying surface area by tissue depth measured from replicate decalcified pieces of M. capitata. Extract concentrations were determined as g dried extract ml-1 of coral tissue. Extracts were re-suspended to these concentrations in 2:1 dichloromethane:methanol for use in disk diffusion assays.

Bacterial strains tested: The strains used for our antibacterial assays were selected as model systems to represent a range of potential bacterial pathogens from the marine environment, including known coral pathogens (Aurantimonas coralicida, Serratia marcescens, Vibrio coraliilyticus, Vibrio shiloi) and human enteric bacteria that have the potential to enter near-shore waters and can survive in the marine environment (Yersinia enterocolitica), and strains previously isolated from the surfaces of Hawaiian corals (Klebsiella pneumonia, Vibrio agarivorans) (Gochfeld & Aeby 2008). In addition, bacterial strains isolated from surfaces of Montipora capitata (OCN001, OCN002, OCN003, OCN008, O1-1, O2-8, O2-12, R1-13, R5-5, R5-13, R5-29) by Dr. Sean Callahan’s lab at University of Hawaii were also used. Two of these, OCN002 and OCN008, are pathogens associated with Montipora White Syndrome (Ushijima et al. 2012, 2014).

Disk Diffusion Assay (Gochfeld et al. 2012): Extracts from orange and red colonies (n = 4 replicates of each color morph) were added to 4-mm diameter paper disks at natural volumetric concentrations. Assays were run on triplicate petri plates. Twenty-four hour bacterial cultures were plated onto appropriate medium, and disks impregnated with the organic extracts were placed on the plate. Following a 24-hr incubation period, the zones of inhibition surrounding each disk were measured.

Processing Description

Data represent mean zones of inhibition in millimeters for the three replicate disks of each sample against each bacterium.